{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:05:54Z","timestamp":1760101554781,"version":"build-2065373602"},"reference-count":47,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2025,7,17]],"date-time":"2025-07-17T00:00:00Z","timestamp":1752710400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia, Portugal (FCT), contract program FCT Mobility","award":["1300556293\/2024-25","UI\/BD\/150854\/2021"],"award-info":[{"award-number":["1300556293\/2024-25","UI\/BD\/150854\/2021"]}]},{"name":"FCT","award":["1300556293\/2024-25","UI\/BD\/150854\/2021"],"award-info":[{"award-number":["1300556293\/2024-25","UI\/BD\/150854\/2021"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>This study discloses the noncovalent immobilization of a bienzyme cascade composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto magnetically responsive polyamide microparticles (PA MPs). Porous PA6, PA4, and PA12 MPs containing iron fillers were synthesized via activated anionic ring-opening polymerization in suspension, alongside neat PA6 MPs used as a reference. Four hybrid catalytic systems (GOx\/HRP@PA) were prepared through sequential adsorption of HRP and GOx onto the various PA MP supports. The initial morphologies of the supports and the hybrid biocatalysts were characterized by SEM, followed by evaluation of the catalytic performance using a two-step glucose oxidation cascade process. Among all systems, the GOx\/HRP@PA4-Fe complex exhibited the highest activity, being approximately 1.5 times greater than the native enzyme dyad, followed by the PA6-supported system with slightly inferior performance. All systems obeyed Michaelis\u2013Menten kinetics, with the immobilized cascades displaying higher K\u2098 and V\u2098\u2090\u2093 values than the non-immobilized enzyme pair while maintaining comparable catalytic efficiencies, CE (CE = kcat\/K\u2098). Subsequently, the immobilized and native enzyme systems were employed for the polymerization of aniline. According to UV\u2013VIS, complete monomer conversion was achieved within 24 h for selected catalysts, and FTIR analysis confirmed the formation of polyaniline in the emeraldine base form without the use of template molecules. These findings highlight the potential of Fe-containing polyamide microparticles as efficient supports for the sustainable, enzyme-mediated synthesis of intrinsically conductive aromatic polymers.<\/jats:p>","DOI":"10.3390\/molecules30143003","type":"journal-article","created":{"date-parts":[[2025,7,17]],"date-time":"2025-07-17T10:33:47Z","timestamp":1752748427000},"page":"3003","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Noncovalently Immobilized Glucose Oxidase\/Horseradish Peroxidase Cascade on Polyamide Supports for Eco-Friendly Polyaniline Synthesis"],"prefix":"10.3390","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1698-553X","authenticated-orcid":false,"given":"Nadya V.","family":"Dencheva","sequence":"first","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6342-1967","authenticated-orcid":false,"given":"Joana F.","family":"Braz","sequence":"additional","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]},{"given":"Sofia A.","family":"Guimar\u00e3es","sequence":"additional","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9057-9380","authenticated-orcid":false,"given":"Zlatan Z.","family":"Denchev","sequence":"additional","affiliation":[{"name":"IPC\u2014Institute for Polymers and Composites, University of Minho, 4800-056 Guimar\u00e3es, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2097","DOI":"10.1021\/cr0002590","article-title":"Polymer synthesis by in vitro enzyme catalysis","volume":"101","author":"Gross","year":"2001","journal-title":"Chem. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Zavada, S.R., Battsengel, T., and Scott, T.F. (2016). Radical-Mediated Enzymatic Polymerizations. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17020195"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1007\/s11224-016-0861-3","article-title":"Green polymer chemistry: New methods of polymer synthesis using renewable starting materials","volume":"28","author":"Kobayashi","year":"2016","journal-title":"Struct. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1021\/ja01145a063","article-title":"Chain Chain reactions induced by enzymic systems","volume":"73","author":"Parravano","year":"1951","journal-title":"J. Am. Chem. Soc."},{"key":"ref_5","unstructured":"Kobayashi, S. (2014). Enzymatic Polymerization. Encyclopedia of Polymer Science and Technology, John Wiley & Sons. [4th ed.]."},{"key":"ref_6","first-page":"1796","article-title":"Enzyme-catalyzed atom transfer radical polymerization","volume":"34","author":"Wang","year":"2022","journal-title":"Prog. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e55437","DOI":"10.1002\/app.55437","article-title":"Laccase-mediated polymerization of tannins from a pine bark extract: Toward an eco-friendly valorization of forest wastes","volume":"141","author":"Vera","year":"2024","journal-title":"J. Appl. Polym. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Athanasiou, P.E., Gkountela, C.I., Patila, M., Fotiadou, R., Chatzikonstantinou, A.V., Vouyiouka, S.N., and Stamatis, H. (2024). Laccase-mediated oxidation of phenolic compounds from wine lees extract towards the synthesis of polymers with potential applications in food packaging. Biomolecules, 14.","DOI":"10.3390\/biom14030323"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"16578","DOI":"10.1039\/C5NR05716G","article-title":"Microgel coating of magnetic nanoparticles via bienzyme-mediated free-radical polymerization for colorimetric detection of glucose","volume":"7","author":"Wu","year":"2015","journal-title":"Nanoscale"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.jiec.2016.03.003","article-title":"High-performance supercapacitors based on polyaniline\u2013graphene nanocomposites: Some approaches, challenges and opportunities","volume":"36","author":"Chauhan","year":"2016","journal-title":"J. Ind. Eng. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1039\/dc9898800317","article-title":"Polyanilines: A novel class of conducting polymers","volume":"88","author":"MacDiarmid","year":"1989","journal-title":"Faraday Discuss. Chem. Soc."},{"key":"ref_12","unstructured":"Mozafari, M., and Singh Chauhan, N.P. (2019). Synthetic route of PANI (II): Enzymatic method. Fundamentals and Emerging Applications of Polyaniline, Elsevier."},{"key":"ref_13","unstructured":"Mark, H.F. (2014). Conducting polymers: Polyaniline. Encyclopedia of Polymer Science and Technology, John Wiley & Sons. [4th ed.]."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.enzmictec.2013.12.008","article-title":"The use of Trametes versicolor laccase for the polymerization of aniline in the presence of vesicles as templates","volume":"55","author":"Junker","year":"2014","journal-title":"Enzym. Microb. Technol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Walde, P., Kashima, K., and \u0106iri\u0107-Marjanovi\u0107, G. (2019). Synthesizing polyaniline with laccase\/O2 as catalyst. Front. Bioeng. Biotechnol., 7.","DOI":"10.3389\/fbioe.2019.00165"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.2478\/s11696-013-0307-y","article-title":"Preparation of aqueous polyaniline\u2212vesicle suspensions with class III peroxidases. Comparison between horseradish peroxidase isoenzym C and soybean peroxidase","volume":"67","author":"Junker","year":"2013","journal-title":"Chem. Pap."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6478","DOI":"10.1039\/c2ra20566a","article-title":"Mechanistic aspects of the horseradish peroxidase-catalysed polymerisation of aniline in the presence of AOT vesicles as templates","volume":"2","author":"Junker","year":"2012","journal-title":"RSC Adv."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1016\/j.electacta.2017.11.133","article-title":"Superior capacitive properties of polyaniline produced by a one-pot peroxidase\/H2O2-triggered polymerization of aniline in the presence of AOT vesicles","volume":"258","author":"Junker","year":"2017","journal-title":"Electrochim. Acta"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5071","DOI":"10.1007\/s11696-021-01620-z","article-title":"Application of an enzymatic cascade reaction for the synthesis of the emeraldine salt form of polyaniline","volume":"75","author":"Kurisu","year":"2021","journal-title":"Chem. Pap."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Arana-Pe\u00f1a, S., Carballares, D., Morellon-Sterlling, R., Berenguer-Murcia, \u00c1., Alc\u00e1ntara, A.R., Rodrigues, R.C., and Fernandez-Lafuente, R. (2021). Enzyme co-immobilization: Always the biocatalyst designers\u2019 choice\u2026or not?. Biotechnol. Adv., 51.","DOI":"10.1016\/j.biotechadv.2020.107584"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Zdarta, J., Meyer, A.S., Jesionowski, T., and Pinelo, M. (2018). A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility. Catalysts, 8.","DOI":"10.3390\/catal8020092"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Xu, K., Chen, X., Zheng, R., and Zheng, Y. (2020). Immobilization of multienzymes on support materials for efficient biocatalysis. Front. Bioeng. Biotechnol., 8.","DOI":"10.3389\/fbioe.2020.00660"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1254","DOI":"10.1016\/j.cej.2019.05.141","article-title":"Recent progress in multienzymes co-immobilization and multienzyme system applications","volume":"373","author":"Ren","year":"2019","journal-title":"Chem. Eng. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1332","DOI":"10.1021\/jz1002007","article-title":"Channeling by proximity: The catalytic advantages of active site colocalization using Brownian dynamics","volume":"1","author":"Bauler","year":"2010","journal-title":"J. Phys. Chem. Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1038\/nchem.2459","article-title":"Substrate channeling as an approach to cascade reactions","volume":"8","author":"Wheeldon","year":"2016","journal-title":"Nat. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1038\/nnano.2014.100","article-title":"Multienzyme complexes on DNA scaffolds capable of substrate channeling with an artificial swinging arm","volume":"9","author":"Fu","year":"2014","journal-title":"Nat. Nanotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8658","DOI":"10.1021\/nn402823k","article-title":"Origins of activity enhancement in enzyme cascades on scaffolds","volume":"7","author":"Idan","year":"2013","journal-title":"ACS Nano"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"13982","DOI":"10.1038\/ncomms13982","article-title":"Proximity does not contribute to activity enhancement in the glucose oxidase\u2013horseradish peroxidase cascade","volume":"7","author":"Zhang","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5161","DOI":"10.1021\/acscatal.6b01302","article-title":"Role of dimension and spatial arrangement on the activity of biocatalytic cascade reactions on scaffolds","volume":"6","author":"Chado","year":"2016","journal-title":"ACS Catal."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Braz, J.F., Dencheva, N.V., Malfois, M., and Denchev, Z.Z. (2023). Synthesis of novel polymer-assisted organic-inorganic hybrid nanoflowers and their application in cascade biocatalysis. Molecules, 28.","DOI":"10.3390\/molecules28020839"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Braz, J.F., Dencheva, N.V., Tohidi, S.D., and Denchev, Z.Z. (2023). Fast, multiple-use optical biosensor for point-of-care glucose detection with mobile devices based on bienzyme cascade supported on polyamide 6 microparticles. Polymers, 15.","DOI":"10.3390\/polym15132802"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3669","DOI":"10.1016\/S0021-9258(18)34832-4","article-title":"The horseradish peroxidase-catalyzed oxidation of 3,5,3\u2032,5\u2032-tetramethylbenzidine\u2014Free radical and charge-transfer complex intermediates","volume":"257","author":"Josephy","year":"1982","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/S0022-0728(97)00305-7","article-title":"Resonance Raman and absorption spectroscopic studies on the electrochemical oxidation processes of 3,3\u2032,5,5\u2032-tetramethylbenzidine","volume":"436","author":"Misono","year":"1997","journal-title":"J. Electroanal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/j.snb.2006.06.006","article-title":"Optimizing the ratio of horseradish peroxidase and glucose oxidase on a bienzyme electrode: Comparison of a theoretical and experimental approach","volume":"122","author":"Mackey","year":"2007","journal-title":"Sens. Actuators B Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1021\/cs401009z","article-title":"Design and analysis of enhanced catalysis in scaffolded multienzyme cascade reactions","volume":"4","author":"Lin","year":"2014","journal-title":"ACS Catal."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2104884","DOI":"10.1002\/advs.202104884","article-title":"Multimodal enzyme-carrying suprastructures for rapid and sensitive biocatalytic cascade reactions","volume":"9","author":"Jo","year":"2022","journal-title":"Adv. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"21205","DOI":"10.1039\/C7RA02291C","article-title":"Magnetic metal\u2013organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis","volume":"7","author":"Chen","year":"2017","journal-title":"RSC Adv."},{"key":"ref_38","first-page":"411","article-title":"H. The kinetics of enzyme cascade systems. General kinetics of enzyme cascade","volume":"173","author":"Hemker","year":"1969","journal-title":"Proc. R. Soc. London. Ser. B Biol. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1080\/1023666X.2012.658651","article-title":"The chemical and physical characterizations of aniline-co-3-methyl thiophene copolymer synthesized by a new oxidant","volume":"17","author":"Can","year":"2012","journal-title":"Int. J. Polym. Anal. Charact."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1196","DOI":"10.1002\/er.3920","article-title":"Storage and evolution of hydrogen in acidic medium by polyaniline","volume":"42","author":"Padmapriya","year":"2017","journal-title":"Int. J. Energy Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"123679","DOI":"10.1016\/j.jssc.2022.123679","article-title":"Polyaniline and its composites engineering: A class of multifunctional smart energy materials","volume":"317","author":"Goswami","year":"2022","journal-title":"J. Solid State Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"27404","DOI":"10.1039\/C4RA02296C","article-title":"Doping-induced detection and determination of propellant grade hydrazines by a kinetic spectrophotometric method based on nano and conventional polyaniline using halide ion releasing additives","volume":"4","author":"Subramanian","year":"2014","journal-title":"RSC Adv."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1016\/j.polymer.2018.04.080","article-title":"One-pot low temperature synthesis and characterization of hybrid poly(2-pyrrolidone) (PA4) microparticles suitable for protein immobilization","volume":"145","author":"Dencheva","year":"2018","journal-title":"Polymer"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1002\/mame.201500194","article-title":"One-step in situ synthesis of polyamide microcapsules with inorganic payload and their transformation into responsive thermoplastic composite materials","volume":"301","author":"Dencheva","year":"2016","journal-title":"Macromol. Mater. Eng."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e51784","DOI":"10.1002\/app.51784","article-title":"Synthesis and properties of neat, hybrid, and copolymeric polyamide 12 microparticles and composites on their basis","volume":"139","author":"Dencheva","year":"2022","journal-title":"J. Appl. Polym. Sci."},{"key":"ref_46","unstructured":"Kohan, M.I. (1995). Nylon Plastics Handbook, Hanser."},{"key":"ref_47","unstructured":"Novitsky, T.F. (2009). Random and Block Copolymers of PA12. [Ph.D. Dissertation, University of Southern Mississippi]. Available online: https:\/\/aquila.usm.edu\/dissertations\/1092."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/14\/3003\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:11:18Z","timestamp":1760033478000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/14\/3003"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,17]]},"references-count":47,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["molecules30143003"],"URL":"https:\/\/doi.org\/10.3390\/molecules30143003","relation":{},"ISSN":["1420-3049"],"issn-type":[{"type":"electronic","value":"1420-3049"}],"subject":[],"published":{"date-parts":[[2025,7,17]]}}}